Temporal variability in stable isotope ratios of C and N in the vibrissa of captive and wild adult South American sea lions Otaria byronia: More than just diet shifts

We analyzed the δ¹³C and δ¹⁵N values in the vibrissae of captive adult breeding South American sea lions (Otaria byronia) fed at a constant diet and then used this information to analyze the change in stable isotope values along the vibrissae from wild individuals. The overall diet‐to‐vibrissa discrimination factor of the captive animals was 3.0‰ ± 0.1‰ for δ¹³C and 3.6‰ ± 0.1‰ for δ¹⁵N, but the stable isotope ratios fluctuated periodically despite constant diet. The δ¹³C and δ¹⁵N values of the captive male declined at the end of the breeding season, whereas the δ¹³C values of the female increased during the central part of pregnancy and the δ¹⁵N values peaked during lactation. The δ¹³C and δ¹⁵N values of adult wild specimens also fluctuated periodically and vibrissae growth rate (0.15 mm/d in both sexes) was slightly lower than in captivity (0.17 mm/d), assuming an annual periodicity for oscillations. Similarities in the amplitude of the cycles of captive and wild males suggested that fasting was probably the main source of periodic variability in the δ¹⁵N of wild males, whereas pregnancy and lactation were probably the main source of periodic variability for the δ¹³C of wild females.     

Stable isotope records of sei whale baleens from Chilean Patagonia as archives for feeding and migration behavior

Carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotope variations in baleen plates of sei whales (Balaenoptera borealis) stranded after a mass mortality event in Chilean Patagonia were investigated to assess potential dietary and migratory patterns. Carbon and nitrogen isotope ratios of seven baleens from six individuals were analyzed. The δ¹³C values ranged from ? 19.1 to ? 15.9‰ and the δ¹⁵N values from 8.7 to 15.4‰. Variations of up to 2.9‰ for δ¹³C and 5.3‰ for δ¹⁵N were observed within one baleen. Carbon and nitrogen isotope records of each baleen were significantly correlated and showed recurring oscillations confirmed by wavelet analyses. Oscillations slightly differed in periodicity indicating variable baleen growth rates between 10.0 and 16.5 cm/year. Food sources of the whales are discussed in terms of available isotope data for potential prey taxa and potential migratory behavior on the basis of latitudinal isotope gradients of particulate organic matter. Cyclicity could be explained by regular migrations of the sei whales from subtropical calving areas to high‐latitude foraging grounds. δ¹⁵N records of baleens differed between individuals eventually pointing to diverse feeding and migratory preferences among sei whale individuals.     

Using stable isotopes to understand changes in ringed seal foraging ecology as a response to a warming environment

Trends toward increased temperatures, reduced sea ice extent, and longer open water seasons have resulted in changing Arctic ecosystem dynamics. Expected changes include shifts in distribution and abundance of prey species for seabirds and marine mammals. Using stable isotope analysis, we studied spatial and interannual variation in ringed seal (Pusa hispida) feeding ecology in Hudson Bay in relation to environmental variables, between 2003 and 2010. Ringed seal muscle and hair samples collected from Arviat and Sanikiluaq, Nunavut, were analyzed for stable isotope ratios of nitrogen (δ¹⁵N) and carbon (δ¹³C). Seals from western Hudson Bay (Arviat) had higher δ¹⁵N and lower δ¹³C than seals from eastern Hudson Bay (Sanikiluaq), and stable isotope ratios varied interannually within each region. Peak δ¹⁵N occurred in years with spring air temperatures between approximately ?5°C and ?2°C. This temperature range was characteristic of warm years in western Hudson Bay and cool years in eastern Hudson Bay. We hypothesize that the high δ¹⁵N observed in ringed seals is indicative of greater importance of capelin (Mallotus villosus) in ringed seal diet. A comparison of ringed seal isotopic niche widths indicated greater dietary differences between western and eastern Hudson Bay with warming, suggesting a possible ecological divergence related to climate change.     

Intra‐lake stable isotope ratio variation in selected fish species and their possible carbon sources in Lake Kyoga (Uganda): implications for aquatic food web studies

The stable isotopes of nitrogen (δ¹⁵N) and carbon (δ¹³C) provide powerful tools for quantifying trophic relationships and carbon flow to consumers in food webs; however, the isotopic signatures of organisms vary within a lake. Assessment of carbon and nitrogen isotopic signatures in a suite of plants, invertebrates, and fishes in Lake Kyoga, indicated significant variation between two sites for δ¹³C (paired t = 6.305; df = 14, P < 0.001 and δ¹⁵N paired t = 1.292; df = 14; P < 0.05). The fish fauna in Bukungu was generally more ¹³C enriched (mean δ¹³C = –16.37 ± 1.64‰) than in Iyingo (mean δ¹³C = –20.80 ± 2.41‰) but more δ¹⁵N depleted (mean δ¹⁵N = 5.57 ± 0.71‰) than in Iyingo (mean δ¹⁵N = 6.92 ± 0.83‰). The simultaneous shifts in phytoplankton and consumer signatures confirmed phytoplankton as the major source of carbon for the food chain leading to fish. Limited sampling coverage within lakes may affect lake wide stable isotope signatures, and the same error is transferred into trophic position estimation. Consideration of potential intra‐lake spatial variability in isotope ratios and size is essential in evaluating the spatial and trophic structure of fish assemblages.     

Diet composition and seasonal feeding patterns of a freshwater ringed seal (Pusa hispida saimensis)

The Saimaa ringed seal (Pusa hispida saimensis) is one of the few freshwater seal populations worldwide. The major conservation issue of this critically endangered population is bycatch mortality. We used digestive tract content and stable isotopes (δ¹³C and δ¹⁵N) to estimate the diet and seasonal feeding patterns for gaining better understanding of the seals feeding habits and potential conservation implications. The diet was similar across age groups. Altogether 15 fish species were identified and the most important were smelt (Osmerus eperlanus), ruff (Gymnocephalus cernuus), perch (Perca fluviatilis), vendace (Coregonus albula) and cyprinids. The high δ¹⁵N values suggested that the seals lose weight during winter and spring. Additionally the drop in δ¹⁵N values indicated that pups start to recover from postweaning stress and gain weight around the age of 6 mo. The isotope values differed regionally, which emphasizes that samples from consumers and prey should be collected from the same regions to improve interpretation of the stable isotopic results. Overall, diet composition suggests minimal to nonexistent competition with commercial or recreational fishing. However, observed weight loss of pups during summer may be related to higher risk of bycatch and this should be taken into account when planning temporal fishing closures.     

Effect of lipid removal on carbon and nitrogen stable isotope ratios in crustacean tissues

The analysis of tissue's naturally occurring stable carbon and nitrogen isotope ratios is a useful tool to delineate trophic relationships. However, the interpretation of δ¹³C and δ¹⁵N is complicated by the influence of multiple factors such as the tissue-specific lipid content. The aim of this work was to evaluate the effects of lipid extraction on δ¹³C and δ¹⁵N compositions in muscle, hepatopancreas and gonads of a marine decapod crustacean, the spider crab Maja brachydactyla. Samples were analyzed for stable isotopes before and after lipid removal, using a derived Soxhlet extraction method. Differences in δ¹³C and δ¹⁵N were measured among tissues before and after treatment. Lipid extraction of muscle did not have a significant effect on either δ¹³C or δ¹⁵N. By contrast, ecologically significant shifts for both carbon and nitrogen stable isotopes ratios (+ 2.9 ± 0.8‰ for δ¹³C, and + 1.2 ± 0.7‰ for δ¹⁵N) were noticed in the hepatopancreas. In regard to gonads, lipid extraction led to a shift only on δ¹³C (+ 1.3 ± 0.3‰). Finally, the derived Soxhlet extraction method removed the lipid influence for δ¹³C, and had an effect on δ¹⁵N composition for lipid-rich samples. We recommend this treatment for carbon stable isotope studies on decapod crustacean lipid-rich tissues.     

Temperature and diet affect carbon and nitrogen isotopes of fish muscle: can amino acid nitrogen isotopes explain effects?

Stable isotope ratios of carbon (δ¹³C) and nitrogen (δ¹⁵N) are widely used in food-web studies to determine trophic positioning and diet sources. However in order to accurately interpret stable isotope data the effects of environmental variability and dietary composition on isotopic discrimination factors and tissue turnover rates must be validated. We tested the effects of temperature and diet on tissue turnover rates and discrimination of carbon and nitrogen isotopes in an omnivorous fish, black bream (Acanthopagrus butcheri). Fish were raised at 16 °C or 23 °C and fed either a fish-meal or vegetable feed to determine turnover rates in fish muscle tissue up to 42 days after exposure to experimental treatments. Temperature and diet affected bulk tissue δ¹⁵N turnover and discrimination factors, with increased turnover and smaller discrimination factors at warmer temperatures. Fish reared on the vegetable feed showed greater bulk tissue δ¹⁵N changes and larger discrimination factors than those reared on a fish-meal feed. Temperature and diet affected bulk tissue δ¹³C values, however the direction of effects among treatments changed. Analyses of δ¹⁵N values of individual amino acids found few significant changes over time or treatment effects, as there was large variation at the individual fish level. However glutamic acid, aspartic acid and leucine changed most over the experiment and results mirrored those of treatment effects in bulk δ¹⁵N tissue values. The results demonstrate that trophic discrimination for δ¹⁵N and δ¹³C can be significantly different than those typically used in food-web analyses, and effects of diet composition and temperature can be significant. Precision of compound-specific isotope analyses (0.9‰) was larger than our effect size for bulk δ¹⁵N diet effects (0.7‰), therefore future experimental work in this area will need to establish a large effect size in order to detect significant differences. Our results also suggest that compound-specific amino acid δ¹⁵N may be useful for determining essential and non-essential amino acids for different animals.     

Stable isotope enrichment in laboratory ant colonies: effects of colony age,metamorphosis, diet,and fat storage

Ecologists use stable isotopes to infer diets and trophic levels of animals in food webs, yet some assumptions underlying these inferences have not been thoroughly tested. We used laboratory‐reared colonies of Solenopsis invicta Buren (Hymenoptera: Formicidae: Solenopsidini) to test the effects of metamorphosis, diet, and lipid storage on carbon and nitrogen stable isotope ratios. Effects of metamorphosis were examined in ant colonies maintained on a control diet of domestic crickets and sucrose solution. Effects of a diet shift were evaluated by adding a tuna supplement to select colonies. Effects of lipid content on stable isotopes were tested by treating worker ants with polar and non‐polar solvents. δ¹³C and δ¹⁵N values of larvae, pupae, and workers were measured by mass spectrometry on whole‐animal preparations. We found a significant effect of colony age on δ¹³C, but not δ¹⁵N; larvae, pupae, and workers collected at 75 days were slightly depleted in ¹³C relative to collections at 15 days (Δδ¹³C = ?0.27‰). Metamorphosis had a significant effect on δ¹⁵N, but not δ¹³C; tissues of each successive developmental stage were increasingly enriched in ¹⁵N (pupae, +0.5‰; workers, +1.4‰). Availability of tuna resulted in further shifts of about +0.6‰ in isotope ratios for all developmental stages. Removing fat with organic solvents had no effect on δ¹³C, but treatment with a non‐polar solvent resulted in enriched δ¹⁵N values of +0.37‰. Identifying regular patterns of isotopic enrichment as described here should improve the utility of stable isotopes in diet studies of insects. Our study suggests that researchers using ¹⁵N enrichment to assess trophic levels of an organism at different sites need to take care not to standardize with immature insect herbivores or predators at one site and mature ones at another. Similar problems may also exist when standardizing with holometabolous insects at one site and spiders or hemimetabolous insects at another site.     

Differences in δ15N and δ13C between embryonic and maternal tissues of the ovoviviparous bluntnose sixgill shark Hexanchus griseus

Stable nitrogen (δ¹⁵N) and carbon (δ¹³C) isotope ratios from muscle, liver and yolk were analysed from the mother and embryos of an ovoviviparous shark, Hexanchus griseus. Embryonic liver and muscle had similar δ¹⁵N and δ¹³C ratios or were depleted in heavy isotopes, compared to the same maternal somatic and reproductive yolk tissues, but no relationship existed between δ¹⁵N or δ¹³C and embryo length, as expected, because a switch to placental nourishment is lacking in this species. This study expands the understanding of maternal nourishment and embryonic stable isotope differences in ovoviviparous sharks.     

Consumer‐resource stoichiometry as a predictor of trophic discrimination (Δ13C, Δ15N) in aquatic invertebrates

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Within-lake variability in carbon and nitrogen stable isotope signatures

1. We assessed spatial and temporal variation in carbon and nitrogen isotopic signatures in different compartments of a single lake ecosystem. Stable isotope analyses were made on samples of particulate organic matter (POM), zooplankton, periphyton, macrophytes, macroinvertebrates and fish collected from several locations throughout the ice‐free period. 2. No spatial variation in δ¹³C or δ¹⁵N values was found for pelagic samples of POM and zooplankton. However, pelagic δ¹⁵N signatures increased steadily through the summer resulting in an almost 6‰ average increase in POM and zooplankton. A concurrent decrease in epilimnetic nitrate concentrations suggested that the increase in δ¹⁵N of POM and zooplankton could have resulted from a progressive ¹⁵N‐enrichment of the available inorganic nitrogen pool as the size of this pool was reduced. 3. Significant spatial variation in isotopic ratios was observed within littoral and profundal communities. Some spatial differences were likely related to lake‐specific characteristics, such as a major inlet and a small harbour area and some were interconnected with temporal events. 4. Marked differences between spring and autumn δ¹⁵N and δ¹³C values of fish at one site probably reflected a spring spawning immigration from a larger downstream lake and also indicated limited dispersal of these immigrants. 5. Our results indicate that restricted sampling of ecosystem components from lakes may provide misleading single values for the isotope end members needed for quantitative uses of stable isotopes in mixing models and for estimating trophic position. Hence we strongly advise that studies of individual lakes, or multiple lake comparisons, that utilise stable isotope analyses should pay more attention to potential within lake spatial and temporal variability of isotope ratios.     

Retrospective analysis of bottlenose dolphin foraging: A legacy of anthropogenic ecosystem disturbance

We used stable isotope analysis to investigate the foraging ecology of coastal bottlenose dolphins (Tursiops truncatus) in relation to a series of anthropogenic disturbances. We first demonstrated that stable isotopes are a faithful indicator of habitat use by comparing muscle isotope values to behavioral foraging data from the same individuals. δ¹³C values increased, while δ³⁴S and δ¹⁵N values decreased with the percentage of feeding observations in seagrass habitat. We then utilized stable isotope values of muscle to assess temporal variation in foraging habitat from 1991 to 2010 and collagen from tooth crown tips to assess the time period 1944 to 2007. From 1991 to 2010, δ¹³C values of muscle decreased while δ³⁴S values increased indicating reduced utilization of seagrass habitat. From 1944 to 1989 δ¹³C values of the crown tip declined significantly, likely due to a reduction in the coverage of seagrass habitat and δ¹⁵N values significantly increased, a trend we attribute to nutrient loading from a rapidly increasing human population. Our results demonstrate the utility of using marine mammal foraging habits to retrospectively assess the extent to which anthropogenic disturbance impacts coastal food webs.     

Stable isotope fractionation between maternal and embryo tissues in the Bonnethead shark (<Emphasis Type="Italic">Sphyrna tiburo</Emphasis>)

Evaluating tissue fractionation between mothers and their offspring is fundamental for informing our interpretation of stable isotope values in young individuals and can provide insight into the dynamics of maternal provisioning. The objectives of this study were to investigate the isotopic relationships between maternal reproductive (i.e., yolk, yolk-sac placenta) and somatic tissues (i.e., muscle and liver) relative to embryos in the Bonnethead Shark Sphyrna tiburo, to evaluate the fractionation of stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotopes between these tissues. Additionally, we examined intra-uterine variability in the isotopic relationships to ascertain whether this species may exhibit variable nutrient allocation. Embryos showed similar magnitudes of enrichment in ¹³C (i.e., Δδ¹³C, difference between adult and embryo) relative to adult tissues (Δδ¹³C?=?~1.0‰). However, embryos were depleted in ¹⁵N relative to adult muscle tissues (Δδ¹⁵N?=??1.0‰), a finding that contrasts Δδ¹⁵N values reported for other placentotrophic sharks. Embryo-muscle Δδ¹⁵N was correlated with length, supporting the contention that the magnitude of enrichment between embryonic and maternal tissues results from the shift from yolk to placental feeding. Embryo δ¹⁵N and Δδ¹⁵N values showed significant intra-uterine variability; a result not observed for δ¹³C and Δδ¹³C values. The contrasting patterns in fractionation among placentotrophic sharks highlight the importance of evaluating these relationships across elasmobranch taxa with consideration for different tissues, reproductive strategies and stages of gestation. The divergent findings support future evaluation of stable isotope relationships between mothers and offspring for purposes of estimating inherent isotopic variability and how this variability may inform physiological and dietary mechanisms.     

Stable Isotope Trophic Shifts in White-Tailed Deer

Abstract Differences between diet and tissue isotope values known as trophic shifts (Δδ¹³C and Δδ¹⁵N) occur during digestion and assimilation of consumed food. Consideration of trophic shifts is essential when using stable isotopes for dietary reconstruction but has received little attention for cervids. Therefore, our purpose was to determine C and N trophic shifts in tissues of captive white-tailed deer (Odocoileus virginianus) fed corn and alfalfa in known amounts over a 4-month period. Antler has also received limited consideration for use in dietary reconstruction, thus, we analyzed tissue to expose variation among locations along the main beam and between antler components. We collected antler, hair, red blood cells (RBCs), and serum at the end of the feeding trial and analyzed them to determine C (δ¹³C) and N (δ¹⁵N) isotope values. Trophic shifts occurred between diet and all tissues for both isotopes with mean Δδ¹³C = 1.19 ± 2.23% and Δδ¹⁵N = 4.93 ± 0.74%. Antler trophic shifts were greater than those in all other tissues for δ¹³C, whereas antler and RBCs shared similar trophic enrichment over diet but differed from hair and serum for Δδ¹⁵N. Trophic shift values were significantly related to diet in hair and serum for δ¹³C and antler and RBCs for Δδ¹⁵N. Isotope values for antler core and periphery plus antler locations along the main beam did not vary. Antler collagen significantly varied from whole antler for δ¹³C but not δ¹⁵N. Our findings provide mean trophic shift values by tissue that can be used for dietary reconstruction in the study and management of cervids.     

Adapt or die—Response of large herbivores to environmental changes in Europe during the Holocene

Climate warming and human landscape transformation during the Holocene resulted in environmental changes for wild animals. The last remnants of the European Pleistocene megafauna that survived into the Holocene were particularly vulnerable to changes in habitat. To track the response of habitat use and foraging of large herbivores to natural and anthropogenic changes in environmental conditions during the Holocene, we investigated carbon (δ¹³C) and nitrogen (δ¹⁵N) stable isotope composition in bone collagen of moose (Alces alces), European bison (Bison bonasus) and aurochs (Bos primigenius) in Central and Eastern Europe. We found strong variations in isotope compositions in the studied species throughout the Holocene and diverse responses to changing environmental conditions. All three species showed significant changes in their δ¹³C values reflecting a shift of foraging habitats from more open in the Early and pre‐Neolithic Holocene to more forest during the Neolithic and Late Holocene. This shift was strongest in European bison, suggesting higher plasticity, more limited in moose, and the least in aurochs. Significant increases of δ¹⁵N values in European bison and moose are evidence of a diet change towards more grazing, but may also reflect increased nitrogen in soils following deglaciation and global temperature increases. Among the factors explaining the observed isotope variations were time (age of samples), longitude and elevation in European bison, and time, longitude and forest cover in aurochs. None of the analysed factors explained isotope variations in moose. Our results demonstrate the strong influence of natural (forest expansion) and anthropogenic (deforestation and human pressure) changes on the foraging ecology of large herbivores, with forests playing a major role as a refugial habitat since the Neolithic, particularly for European bison and aurochs. We propose that high flexibility in foraging strategy was the key for survival of large herbivores in the changing environmental conditions of the Holocene.     

Isotopic fractionation with morphological change and sexual specificity in the lappet moth Euthrix potatoria

The δ¹⁵N values of adult holometabolous insects exceed those of larvae, but otherwise little information on terrestrial invertebrates has been obtained in food‐web analyses using stable isotope ratios (δ¹⁵N, δ¹³C). Changes in δ¹³C during metamorphosis and differences between males and females have not been examined. We collected the larvae and cocoons of Euthrix potatoria (L.) (Lepidoptera: Lasiocampidae) in the field and used them to assess the species’ isotopic fractionation. Each emerged moth was divided into five body parts. We conducted stable N and C isotope analyses for each body part, as well as for cocoons and exuviae, and also compared stable isotope ratios between sexes. We confirmed δ¹⁵N enrichment through metamorphosis and estimated that δ¹⁵N enrichment is accomplished by the relative concentration of ¹⁵N due to the excretion of copious meconium, which contains abundant ¹⁴N. We also observed changes in δ¹³C values through metamorphosis. Both isotope values tended to change more in males than in females. The proportion of the whole‐adult weight represented by meconium was higher in males than in females, suggesting that high meconium secretion in males contributes to the sexual difference in δ¹⁵N. These phenomena may be common in Holometabola, which require a pupal stage. For more accurate food‐web assessments, it is important to consider stable isotope changes during different life cycles, as well as sexual differences.     

Effects of ethanol storage and lipid and urea extraction on δ15N and δ13C isotope ratios in a benthic elasmobranch,the bluespotted maskray Neotrygon kuhlii

Ethanol storage and lipid and urea extraction had no effect on bluespotted maskray Neotrygon kuhlii muscle δ¹³C values whereas urea‐removal and ethanol storage increased δ¹⁵N values. Results presented here show a significant δ¹⁵N increase post‐urea removal and provide additional support for this approach in future elasmobranch stable‐isotope analysis (SIA) studies. Further experimental work on other elasmobranch species is needed to assess extraction and preservation effects on stable‐isotope (SI) values.     

Turnover rates of nitrogen stable isotopes in the salt marsh mummichog, Fundulus heteroclitus, following a laboratory diet switch

Nitrogen stable isotopes are frequently used in ecological studies to estimate trophic position and determine movement patterns. Knowledge of tissue-specific turnover and nitrogen discrimination for the study organisms is important for accurate interpretation of isotopic data. We measured δ¹⁵ N turnover in liver and muscle tissue in juvenile mummichogs, Fundulus heteroclitus, following a laboratory diet switch. Liver tissue turned over significantly faster than muscle tissue suggesting the potential for a multiple tissue stable isotope approach to study movement and trophic position over different time scales; metabolism contributed significantly to isotopic turnover for both liver and muscle. Nitrogen diet-tissue discrimination was estimated at between 0.0 and 1.2‰ for liver and –1.0 and 0.2‰ for muscle. This is the first experiment to demonstrate a significant variation in δ¹⁵ N turnover between liver and muscle tissues in a fish species.     

Depth variation in isotopic composition of benthic resources and assessment of sculpin feeding patterns in an oligotrophic Alaskan lake

Stable isotopes of carbon (C) and nitrogen (N) are commonly used to track resource flows through lake food webs. However, there remains a weak understanding of the spatial variation in isotopic composition of benthic resources and how this variation affects inference about energy flows among species. Boundary layers at the interface between benthic substrates and the overlying water column restrict diffusion of nutrients and carbon from the water column to benthic algae and may affect the isotopic composition of benthic algae as nutrient and CO₂ concentrations can become locally depleted in the benthic boundary layer. We quantified the variation in C and N stable isotope composition of benthic resources along a depth gradient in a large oligotrophic lake to assess the magnitude of change in stable isotope composition. Snails were increasingly depleted in ¹³C with depth, by about 10 ‰ from 0 to 20 m, while ¹⁵N in snails showed only subtle enrichment over this depth range. Sculpin (Cottas aleuticus) δ ¹³C and δ ¹⁵N signatures did not significantly change with depth and were more enriched in ¹⁵N than would be expected from consumption of snails alone. A comparison of δ ¹³C and δ ¹⁵N values from sculpins relative to shallow and deep snails, and alternative prey (marine-derived salmon resources), within a mixing model suggested sculpins feed selectively on deep grazers in this system in addition to marine-derived resources provided by migrating sockeye salmon. This study illustrates the importance of accounting for depth-related variation in isotope patterns when assessing benthic resource contributions to food webs using stable isotope data.     

Placing butterflies on the map – testing regional geographical resolution of three stable isotopes in Sweden using the monophagus peacock Inachis io

Stable isotope analyses of tissues have been used to help delineate natal regions and routes of migratory animals. The foundations of such studies are isotopic gradients or differences representing geographic regions and habitat used by the organism that are retained in selected tissues. We sampled peacock butterflies Inachis io on a regional level in southern Sweden to study natural variation and the resolving power of the stable isotope method to delineate individuals from known areas on a smaller scale than has typically been used in previous studies. Hydrogen (δD), carbon (δ¹³C) and nitrogen (δ¹⁵N) isotopes were obtained from butterflies at seven different locations in an area of 250×250 km over three years (2002–2004). We found sufficient isotopic differences on this regional scale to delineate approximate origins. Of the three isotopes, deuterium showed good discrimination between sites, carbon isotopes showed weaker differentiation, whereas nitrogen isotopes proved unsuitable for small scale studies in this region due to high and unpredictable variation. We found there was enough variation in δD between years to prevent a general application of the technique to resolve sub‐regional variation. Substantial part of this variation was probably caused by seasonal changes in δD of precipitation. These differences produce significant variation in δD between years in animals having short and variable tissue development times, and are difficult to estimate in natural situations. We conclude that stable isotopes are potentially powerful predictors for studies of migratory butterflies in Europe. However, without good knowledge about the sampled individuals’ previous life‐history, a lot of the natural environmental variation in tissue δD cannot be controlled for. In the case of migratory species, this information is difficult to obtain, making the confidence intervals for prediction of natal areas fairly wide and probably only suitable for longer distance migration.